PROJECT

ColiAlert

How to do a quality control system? ColiAlert!

All laboratory as well as, every machine must have a quality control. Our idea was to use a system based on colorimetric changes to control the quality of the automated extraction robot system. For this, we created ColiAlert. The ColiAlert are bacteria genetically modified that present pink color due the presence of chromoproteins. For this, the biobricks sent to construct a composite that under natural conditions expressed pink, and after extraction procedures, shows yellow color. For this purpose, we decided to use in our project, biobricks provided by the registry. At that stage, it was very important software developed by team-Aalto Helsinki, the BiobrickSeeker. Much time was saved thanks to this tool, which facilitates the search of biobricks sent. Then, were selected chromoproteins available in the registry by Uppsala team, to build our quality control.

It was then possible to construct 3 biobricks. The first (K1565000) is an indicator of cell lysis by the presence of oleic acid. Oleic acid is one of the fatty acids present in the plasmatic membrane. This acid is released in the presence of cell lysis. We built a biobrick with a promoter sensitive to the presence of oleic acid, which stimulates the production of blue chromoprotein for signaling process (Figure 1).

With the same purpose of signaling the process of extracting nucleic acids, two more biobricks (K1565001 and K1565002)were created (Figure 2). In these yellow chromoproteins were used. This chromoprotein is sensitive in the presence of pH changes. Thus, E. coli transformed with either of these two biobricks, grew with pink color due to the neutral pH of LB media. However, with the addition of the solution at acidic pH, the bacteria instantaneously change their color to yellow. This is a reversible process. The same modified genetically engineered bacteria, will change their color from pink to yellow, and from yellow to pink with variations in pH. The process becomes irreversible when cell lysis occurs, causing the ColiAlert only remains yellow. The biobricks constructed it was placed genes that promote that chromoproteins remain at plasmatic membrane. The process of lysis of the membrane, probably affects the structure of chromoproteins or ability to react with the basic or neutral conditions, thus maintaining its original yellow color.

This color variation is shown in the graphs of absorbance shown in figure 3 and 4. Figure 3 is an absorbance spectrum generated by readings from 300 to 680nm at pH 4 and 7. It is possible to see that the samples at neutral pH, having two absorption peaks, while acidic pH has only one, showing the transition of colors. In Figure 4 is possible to observe the change in absorbance obtained from samples of ColiAlert diluted in citrate buffer (0.1M) and phosphate buffer (0.1 M) at different pHs in readings of 500nM.

To try to show that ColiAlert signals the process of extracting nucleic acids correctly, we tested the extraction of nucleic acids only with blood, and blood together with ColiAlert. At the end of procedure, the resulting samples were quantified with NanoDrop (Results in Figure 5). It is possible to note the indirect evidence that in the presence of ColiAlert, the amount of DNA generated was more than twice as high. Figure 6 shows the variation in color and pH obtained after the lysis procedure and figure 7, 8 and 9 shows plates with ColiAlert. And figure 10 shows the process of reversible and irreversible change in coloration.

Figure 3. Curve of the absorption spectrum from 300 to 680nm.

Figure 4. The absorption spectrum of ColiAlert at different pHs (Readings at 500nM).

Figure 5. Quantification of extracted DNA (Nanodrop).

Figure 6. From left to right, ColiAlert after lysis solution, ColiAlert in acidic solution, and ColiAlert in basic solution.

Figure 7. Plate with ColiAlert in LB and the transformation to yellow in the presence of acid solution.

Figure 8. Plate with ColiAlert in LB and after the treatment with acid solution.

Figure 9. Plate with ColiAlert and art with the team name.

Figure 10. Shows the process of reversible and irreversible change in coloration.